Hi there,
I'm isolating cells from spleen tissue for flow cytometry. Last time from a single spleen I got around 40 million cells total but I reckon I can get much more with some refinement. Would appreciate it if someone could look at my protocol and advise on tips to maximise yield. I think the main issue for me is length of time in ACK and wether or not this step needs to be RT/on ice as the last time I think I overlysed the cells. This is the protocol I have. Any advice would be greatly appreciated! Thanks in advance!
Media: RPMI, 10% FBS, 1% Pen/strep
ð Take spleen and place in a 40 µm cell strainer placed in a well of a 3.5 cm dish or well of a 6-well plate.
ð Add 2-3 ml media to the cell strainer and mash spleen through the strainer using the rubber end of a ‘plunger’ from a 5 ml syringe.
ð Rinse the cells through the strainer with an additional 5 ml of media.
ð Gently pipette the cells up and down a few times using a Pasteur pipette, to remove clumps and dissociate the cells fully.
ð Place cells in a 50 ml falcon tube and add media to 10 ml.
ð Pellet cells by centrifugation (300 g for 5 min).
ð Remove supernatant and resuspend cells in 5 ml ACK buffer and mix gently. Wait for 1-2 min until the colour of the cell solution changes (RBC lysis has occurred).
ð Fill the tube up to 15ml with media and pellet the cells (300 g for 5 min).
ð The cell pellet should be white (If it is still pink, repeat the lysis steps with ACK).
ð Resuspend the pellet in 10 ml media and count cells. (Usually 100-200 million cells per spleen). Cells will need to be diluted for accurate counting on the NucleoCounter (~1:100). Keep cells on ice!
Hi David,
I am guessing that these are tissues from mice/rats? I use a very similar method to isolate splenocytes from Porcine spleen tissue, usually to culture, and find it works really well. I'd suggest using cold PBS instead of medium during the isolation process, and then swapping to medium for counting. Use cold lysis buffer as well, but personally I'd avoid keeping things on ice unless absolutely essential for you flow analysis.
The biggest factor in my experience, though, has been the source of the tissue; if the tissue is harvested post-mortem then the method of sacrifice and the time taken to harvest the tissue will both impact the overall composition, yield and viability. Harvesting the spleen as fresh as you can should help to mitigate this. As I generally use my cells for culture experiments, I harvest the tissue under brief terminal anaesthesia, which gives much greater yield and viability, but obviously this may not be available to you.
Hope that's helpful, and good luck!
Joe.
Hi Joe,
Thanks for your detailed answer. Yes I should've said it was spleen from mice and it will be relatively fresh (need to transport it from one end of campus to the other) prior to lysis.
My flow protocol recommends keeping cells on ice throughout and it worked well the last time so will stick with that!
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